Hypertension and dyslipidemia, major risk factors for coronary heart disease, are interrelated. The overall dyslipidemia can contribute to a chronic increase in vascular tone resulting in hypertension. In order to reduce cardiovascular risk, it is important to regulate hypertension as well as dyslipidemia. The modern pharmacological therapy is costly and associated with multiple side-effects resulting in patient non-compliance. Thus there is a need to explore alternative therapies particularly from herbal sources as these are cost-effective and possess minimal side-effects. Viola odorata Linn, commonly known as Sweet Violet in English, belongs to the family Violaceae. It is called Banafsha in Indo-Pakistan. The plant is native to Asia, North Africa and Europe. In Pakistan, Viola odorata is found in the northern areas, such as Nathia Gali, Hazara, Kaghan, Swat and Chitral at the height of 1,500 to 2,000 meters. Its history as a medicinal herb dates back as far as 500 BC, when it was known to be used to relieve pain due to cancer. In the traditional system, it has been used for anxiety, insomnia and lowering blood pressure. Viola odorata contains alkaloid, glycoside, saponins, methyl slicylate, mucilage and vitamin C. The plant has been reported to possess antioxidants and diuretics, but no study has been found regarding its blood pressure-lowering or lipid-lowering activity. In the present investigation, we report the antihypertensive and antidyslipidemic effects of Viola odorata along with possible mechanisms.
RESULTS: The phytochemical analysis of the crude extract of Viola odorata (Vo.Cr) showed the presence of alkaloids, saponins, tannins, phenolics, coumarins and flavonoids. Vo.Cr at 0.1, 0.3 and 1.0 mg/kg induced a percent fall of 15.40 ± 1.43, 27.80 ± 2.37 and 48.60 ± 3.35 (mean ± SEM, n = 5), respectively, in mean arterial pressure (MAP) of rats under anaesthesia. In the isolated guinea pig atrium, the plant extract depressed the force and rate of spontaneous contracting atria with respective EC50 values of 0.39 (0.21-0.75, 95 percent CI; n = 3) and 0.40 mg/ml (0.12-1.38; n = 3), similar to verapamil which inhibited the force and rate of atrial contraction with respective EC50 values of 0.59 (0.47-0.75; n = 6) and 0.94 μM (0.68-1.30; n = 6). When tested on resting tension of endothelium-intact tissue, Vo.Cr caused a concentration-dependent (0.03-3.0 mg/ml) vasoconstriction with maximum of 46.4 ± 2.8 percent (n = 4) of PE (1 μM) maximum contraction followed by complete relaxation at the next higher concentration of 5 mg/ml. In denuded tissues, Vo.Cr also induced vasoconstriction with resultant maximum contraction of 82.6 ± 2.6 percent (n = 4) at 3 mg/kg followed by partial relaxation at the dose of 5 mg/ml. When the vasoconstrictor effect of Vo.Cr in intact tissues was reproduced in the presence of L-NAME (0.1 mM), it was enhanced as 79.6 ± 2.9 percent (n = 4) vs 46.4 ± 2.8 percent (n = 4), while the vasodilator effect was partially inhibited at the higher tested concentration of 5 mg/ml. The vasoconstrictor effect of Vo.Cr, in intact and denuded tissues, was completely blocked when repeated in the presence of phentolamine (1 μM), while verapamil was devoid of any stimulatory effect on the baseline in either tissue. Vo.Cr caused a concentration-dependent relaxation of PE (1 μM)-induced contractions in endothelium-intact and denuded rat aortic preparations with respective EC50 values of 4.98 (3.93-6.30; n = 5) and 10.54 mg/ml (9.10-12.21; n = 4). When the relaxant effect of Vo.Cr was reproduced in intact aortic rings in the presence of L-NAME (0.1 mM), the relaxation curve was partially inhibited with resultant EC50 value of 10.11 mg/ml (6.61-15.43; n = 4) vs 4.98 (3.93-6.30; n = 5). When tested on high K+ (80 mM)-induced contraction, Vo.Cr caused relaxation with EC50 value of 4.38 mg/ml (3.87-4.95; n = 7). Verapamil also inhibited PE-induced contractions in endothelium-intact and denuded rat aortic preparations at similar concentrations with respective EC50 values of 0.41 (0.25-0.66; n = 3) and 0.71 μM (0.40-1.26; n = 3), while it inhibited high K+-induced contractions with EC50 value of 0.18 μM (0.13-0.26; n = 4). Pre-treatment of rat aortic rings with Vo.Cr caused a concentration-dependent (0.03-0.1 mg/ml) rightward shift in the Ca++ concentration-response curves constructed in Ca++- free medium, similar to that caused by verapamil at 0.01-0.03 μM. Vo.Cr (0.03-0.3) mg/ml and verapamil (0.03-0.3) μM suppressed PE (1 μM) peak responses in Ca++ free Kreb’s solution in a concentration-dependent manner. Treatment of animals with Tyloxapol (Triton WR-1339) caused an increase in serum total cholesterol and triglycerides compared to normal controls. Pre-treatment of the rats with Vo.Cr at 300 and 600 mg/kg protected against tyloxapol-induced hypercholesterolemia and hypertriglyceridemia similar to atorvastatin (10 mg/kg). Atherogenic diet significantly increased the serum total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) and atherogenic index, while it decreased high density lipoprotein-cholesterol (HDL-C) as compared to control. Supplementation of atherogenic diet with Vo.Cr (600 mg/kg) and atorvastatin (10 mg/kg) prevented the rise of mean serum TC, LDL-C and atherogenic index. Both the treatments significantly increased HDL-C compared to atherogenic control group. However, there was no effect seen on serum triglycerides and glucose levels. The decrease in body weight was accompanied by slight reduction in daily diet consumption. For acute toxicity study, mice were divided into four groups. First group received normal saline (10 ml/kg), while the other three groups were administered graded doses of 1, 3 and 5 g/kg of Vo.Cr, respectively. All animals showed usual activity in home cages and no mortality or gross behavioural changes such as apathy or aggression were observed for 48 hours.
DISCUSSION: The aqueous-methanolic extract of Viola odorata caused a dose-dependent fall in blood pressure of rats under anaesthesia, which is in accordance with its medicinal use in hypertension. Blood pressure is the product of cardiac output and peripheral resistance; hence, increase in either or both can lead to the development of hypertension. In order to explore the underlying mechanism of action, the plant extract was studied in isolated guinea pig atria and rat aortic preparations. In guinea pig right atria, the crude extract showed negative inotropic and chronotropic effects, similar to that caused by verapamil, a standard Ca++ antagonist, which is known to cause cardiac depression through the inhibition of the slow inward current during the action potential plateau. This indicates that the observed cardiac inhibitory effect of the plant extract might be causing a decrease in cardiac output and ultimately a fall in the blood pressure. To characterise the vasodilator effect, when tested in pre-contracted rat aortic preparations, the plant extract inhibited both high K+ (80 mM) and PE (1 μM)-induced vasoconstriction. Influx of extracellular Ca++ through voltage-dependent channels (VDCs) and receptor-operated channels (ROCs) is caused by high K+ and PE, respectively, resulting in increased intracellular Ca++ which causes contraction. This shows that the plant extract has the ability to block Ca++ influx through both VDCs and ROCs. The Ca++ channel blocking (CCB) activity of the crude extract was further confirmed when it shifted the Ca++ concentration-response curves (CRCs) to the right with the suppression of maximum response. Verapamil, a standard CCB used clinically also caused a rightward shift of the Ca++ CRCs in a dose-dependent manner. In addition to VDCs and ROCs, there is another mechanism of contraction in which Ca++ influx into the cell is guided through the Ca++ release from the internal stores of inositol-1, 4, 5-trisphosphate (IP3)-sensitive sarcoplasmic reticulum. When the control responses of PE were taken in Ca++ free medium, the crude extract in increasing concentrations inhibited the PE-induced peaks, indicating that it is also acting through inhibition of the intracellular Ca++ channels. The results were similar to that of verapamil, suggesting the presence of CCB-like activity in the plant extract which might be responsible for its cardiac inhibitory effect in atrial preparation and the blood pressure lowering effect in anaesthetised rats. Though, additional mechanism cannot be ruled out. The vasodilator effect of the crude extract was further studied in isolated rat aortic preparations for its effect on vascular preparations with intact endothelium. The results showed that vasodilatation caused by Vo.Cr was reduced when the endothelium was removed, indicating some role of endothelium-dependent vasodilator mechanisms. There is strong evidence that endothelium-derived relaxing factor (EDRF) is nitric oxide (NO) synthesised in the endothelium by nitric oxide synthase (NOS) from L-arginine. In order to further study the endothelium-dependent component of the crude extract, when the aortic rings with intact endothelium were pretreated with L-NAME, an inhibitor of NOS, the vasodilator effect was partially inhibited. The degree of relaxation induced by Vo.Cr was almost superimposable on that observed in endothelium-denuded preparations, indicating that the endothelium-dependent relaxation was through NO-dependent pathways. When the relaxant effect of the plant extract was further studied in the presence of atropine, pyrilamine and methysergide, it was found insensitive showing that the vasodilator effect of the plant extract did not involve muscarinic, histaminergic or serotonergic receptors, respectively, which are known to have a role in receptor-mediated NO release from the endothelium. Thus, these results indicate the involvement of other possible NO-dependent pathway(s) like the direct release of NO from the endothelial cells. When the extract was tested on resting tension of endothelium-intact and denuded rat aortic rings, it caused a concentration-dependent vasoconstrictor effect. The maximum contractile effect was achieved at the concentration of 3 mg/ml which was significantly greater in the denuded tissues as compared to that in the intact ones. This again indicates the role of endothelium-derived NO which might have hindered the full expression of contractile effect as it is a potent vasodilator. When the vasoconstrictor effect of the plant extract in intact tissues was reproduced in the presence of L-NAME, the effect was augmented reaching similar to that in the denuded tissues which is in line with our findings regarding the vasodilator effect. The vasoconstrictor effect of Vo.Cr was completely blocked in intact and denuded aortic preparations when reproduced in the tissues pretreated with phentolamine, a standard α-adrenergic antagonist. Despite the observed vasoconstrictor effect in the vascular preparation, the plant extract did not show any hypertensive effect in the intact animal, which could probably be due to some of the endogenous mediators in the whole body blocking this vasoconstrictor effect. Alternatively, the combined presence of the strong vasodilator and cardio suppressant components in the plant extract is not letting the extract to express its vasoconstrictor component in terms of an increase in the blood pressure. Whatever might be the reason, the vasoconstrictor element of the plant extract does not seem to be of any clinical significance, while evaluating its antihypertensive effect. This study is also in line with the earlier reports on Acorus calamus and Orchis mascula possessing a combination of vasoconstrictor and vasodilator components without showing any hypertensive activity when tested in intact animals. Interestingly, plant extract also caused antidyslipidemic effect. In order to study the possible mode of action of the lipid-lowering activity of the plant, two different models were used. Tyloxapol-induced dyslipidemia is a widely used model to explore possible mechanism of lipid lowering drugs. It causes drastic increase in serum triglycerides and cholesterol levels due to increase in hepatic cholesterol synthesis particularly by the increase in HMG Co-A reductase (3-hydroxy-3-methyl-glutaryl Co-A reductase) activity and by the inhibition of lipoprotein lipase responsible for hydrolysis of plasma lipids. The plant extract caused a significant inhibition in the rise of serum triglycerides and cholesterol level, which indicates that inhibition of lipid biosynthesis, might be the possible mechanism of its lipid-lowering action. The atherogenic or high-fat diet-induced dyslipidemia model induces a marked increase in serum total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) and atherogenic index by enhancing intestinal absorption and secretion, and decreasing catabolism of cholesterol. It caused a decrease in high density lipoprotein-cholesterol (HDL-C) but did not affect serum triglyceride (TG). Treatment of the rats receiving atherogenic diet with the extract caused a significant decrease in TC and LDL-C, while increased HDL-C without effecting TG and glucose levels. This may be attributed to the presence of phytochemical constituents like flavonoids and saponins in the plant. Flavonoids are reported to lower LDL-C and increase HDL-C concentrations in hypercholesteremic animals. Saponins have shown to inhibit pancreatic lipase activity in high fat diet-fed mice leading to greater fat excretion due to reduced intestinal absorption of dietary fats. The plant extract also markedly reduced atherogenic index which is considered a better indicator of coronary heart disease risk than individual lipoprotein concentration. The lipid lowering potential of the plant extract was comparable with that of atorvastatin which was used as a positive control in this study and is well known lipid-lowering drug acting via inhibition of HMG Co-A reductase. Atherogenic diet also causes oxidative stress (enzymatic and non-enzymatic) in rats. It, therefore, increases oxidation of LDL-C which plays a key role in genesis of atherosclerosis. Antioxidants are known to effectively prevent this kind of cellular damage. The presence of strong antioxidant activity in the extract may offer additional benefit against oxidative stress caused by high cholesterol. There was a significant increase in the body weights of rats on atherogenic diet compared to control group. The gain in the body weight leading to obesity is an obvious effect of such high fat diets intake. It is worth mentioning that in addition to the beneficial effects of Vo.Cr in hypertension and dyslipidemia, it significantly reduced the body weight, via reducing diet intake, bringing close to the body weights of normal diet-fed rats, thus showing the weight-reducing potential of the plant extract. Obesity is amongst major health issues predisposing people to metabolic diseases such as hypertension and diabetes. According to WHO, by 2015, 2.3 billion human adults would be overweight, while 700 million are expected to be obese. There is poor compliance with conventional weight-management programmes of increasing energy expenditure via physical activity, while drug treatment is often associated with rebound weight gain after the cessation of respective drug therapy. In recent times, evaluation of therapeutic options from natural sources for treating obesity (one of the factors involved in the development of metabolic syndrome), are the focus of interest. In this study, apparently, there was no effect on normal blood glucose level which does not rule out its potential as an anti-diabetic agent, and further studies on diabetic models are required. The antihypertensive, cardio-suppressor, vasodilator, antidyslipidemic and weight reducing properties of Vo.Cr strongly attest its usefulness in metabolic syndrome, particularly if it was shown to possess anti-diabetic activity. Natural products of similar pharmacological profile are widely used in the management of metabolic syndrome.
CONCLUSIONS: In summary, the results of this study show that the crude extract of leaves of Viola odorata exhibited blood pressure-lowering effect in rats under anaesthesia. In the isolated tissue preparations, the extract showed vasorelaxation mediated through inhibition of Ca++ influx via membranous Ca++ channels, its release from intracellular stores and NO-mediated pathways, which possibly explain the fall in BP. The plant also showed reduction in body weight and antidyslipidemic effect which may be due to the inhibition of synthesis and absorption of lipids and antioxidant activities. Thus, this study provides a pharmacologic rationale to the medicinal use of Viola odorata in hypertension and dyslipidemia and may be a good candidate to be developed as antihypertensive and antidyslipidemic medicine, with therapeutic potential in obesity and metabolic syndrome.
Extracted from the research paper ‘Studies on the antihypertensive and antidyslipidemic activities of Viola odorata leaves extract’ authored by Hasan S Siddiqi, Malik H Mehmood, Najeebur Rehman and Anwar H Gilani